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Abstract

In a prospective multicenter phase II trial of radioembolization with yttrium-90 (90Y-RE) in chemorefractory liver-dominant metastatic colorectal cancer (mCRC), we showed
that median survival was 12.6 months (95% CI 7.0–18.3) with 48% of 50 patients achieving
disease control. In this extension retrospective study, we analyzed whether a panel
of biomarkers, known to be associated to an adverse clinical outcome, underwent variations
in CRC liver metastases pre and post 90Y-RE.

Although our analysis was conducted in a very limited number cases, these changes
appear strictly related to the response to 90Y-RE therapy and may deserve further investigation on a larger series of patients.

Keywords:

Introduction

Liver metastases are a significant cause of morbidity and mortality for more than
45% of patients who present with colorectal cancer (CRC) [1]. Although chemotherapy regimens combined with biologic agents have improved the control
of liver metastases, the occurrence of hepatic metastases continues to present a life-limiting
prognosis for most patients with advanced CRC [2] being 5 year survival approximately 11%. In the setting of clinical trials, median
overall survival for unresectable metastases have been extended beyond two years using
combinations including oxaliplatin, irinotecan, capecitabine and biologic agents (bevacizumab,
cetuximab, panitumumab) [3,4]. In parallel with these developments, the application of locally ablative procedures,
such as radiofrequency ablation, are increasingly considered beneficial for patients
with unresectable liver-only disease who present with tumors ≤ 3–4 cm in diameter.
These regional treatments for liver metastases can also be used to consolidate the
treatment response with chemotherapy, in order to further increase the number of patients
eligible for resection [5,6]. Despite these gains, one of the major challenges in advanced CRC are the growing
proportion of patients who continue to present with progressive liver involvement
having exhausted all other therapeutic options.

Radioembolization with yttrium-90 (90Y-RE) and, as recently described, with holmium-166 poly (L-lactic acid) labeled microspheres
(166Ho-PLLA-MS) [7], are therapeutic procedures applied to the liver that allow direct delivery of high-dose
radiation to liver tumors (both primary and metastatic) by means of endovascular catheters,
selectively placed within the hepatic arterial vasculature. 90Y and 166Ho-PLLA-MS (resin or glass) microspheres lodge within the neovascular rim of the tumor(s)
[8,9].

In a multicenter phase II trial conducted in highly chemorefractory liver-dominant
metastatic CRC (mCRC), we showed that 48% (24 of 50) of patients achieved disease
control with a median overall survival of 12.6 months following RE with 90Y-radiolabelled resin microspheres [10]. This finding is consistent with the results from other multicenter evaluations using
90Y-RE in the chemorefractory setting [11]. Up to date, there are no studies which have investigated biomarker expression and
response to 90Y-RE therapy.

It is largely described that the ability to avoid apoptosis is one of the major oncogenic
switches contributing to tumor progression. Among the gene coding apoptosis and cell
proliferation protein regulators, Bcl-2, an antiapopototic protein, survivin, one
of the member of the inhibitor of apoptosis (IAP) protein family and p53 may identify
CRC patients at a higher risk of tumor progression [12-14].

In the present retrospective study which is an extension of our previous one [10], we evaluated whether the expression of these biomarkers may undergo to significant
changes before and after 90Y-RE thus providing predictive information of clinical value.

Methods

Patients and treatment

Between May 2005 and August 2007, 50 patients with unresectable, histologically proven
CRC liver metastases and limited extra-hepatic disease (≤ 3 nodules in the same extra-hepatic
organ each < 3 mm), in progression following standard systemic chemotherapy, were
recruited from four Italian centers in a phase II prospective clinical trial conducted
by the Italian Society of Locoregional Therapy in Oncology (SITILO). Further details
of the treatment planning and patient selection have been outlined in our previous
paper [10]. In brief, patients were required to be between 18 and 75 years of age, have liver
metastases measurable by Response Evaluation Criteria in Solid Tumours (RECIST), adequate
renal function (creatinine < 1.5 7 × normal values or creatinine clearance > 50 mL/minute),
hemopoietic function, WHO or ECOG performance status ≤ 2 and were able to give informed
consent. To be eligible for 90Y-RE, patients were required to have: sufficient liver function; hepatic arterial
anatomy that would enable safe delivery of microspheres to the liver only; liver to
lung shunting of < 20% on a pre-treatment technetium-99m labeled macro-aggregated-albumin
(99mTc-MAA) nuclear scan; and a patent main portal vein. Patients were excluded if they
were pregnant, had evidence of local recurrence of primary disease, inflammatory gastrointestinal
disease or had received prior treatment with hepatic arterial chemotherapy or external
beam radiotherapy to the liver. The median interval between diagnosis of mCRC and
90Y-RE was 17 months (range, 6–71 months). To investigate biomarkers expression and
response to 90Y-RE therapy, liver metastases biopsies were taken 8–21 days prior to 90Y-RE and 2 months post-90Y-RE. Tissue specimens were available from 29 patients pre therapy and 15 patients
post therapy. Samples pre- and post-90Y-RE were concomitantly available in 13 patients.

The study was approved by the Ethical Committee at the Regina Elena Cancer Institute
(N°534; 22/03/05) and a written informed consent was obtained by all patients.

Results were considered positive for survivin when at least 20% of tumor cells, independent
of nuclear or cytoplasmic localization, were immunostained, for p53 when 10% of tumor
cell nuclei were labelled, for Bcl-2 when > 5% of cells showed a cytoplasmic immunoreaction.
Ki-67 proliferation index, based on the median value of our series, was regarded as
high if greater than 50% of the cell nuclei were immunostained. Only well preserved
tumor areas were considered for IHC evaluation. The IHC results were evaluated independently
and in a blinded manner by two investigators (MD, MM).

Statistical analysis

The correlation between biomarkers expression and the response to 90Y-RE was tested by the Pearson Chi-Square test and Mac Nemar test. Significance was
assessed at 5% level (p < 0.05). The SPSS statistical software package version 19.0 was used for analyses
(SPSS, Inc, Chicago, IL, USA).

Results

Of the 50 patients included in the SITILO clinical trial, 29 pre-90Y-RE and 15 post-90Y-RE had sufficient tissue material from their liver metastases for IHC evaluation
of survivin, p53, Bcl-2 and Ki-67. As reported in Table 1, we found that, of the 29 liver metastases analyzed pre-90Y-RE, 24 (77.4%) were survivin positive, 27 (93.1%) p53 positive,11 (37.9%) Bcl-2
positive and 18 (62.5%) presented a high Ki-67 proliferation index (>50%). Of the
15 liver metastases available post-90Y-RE, survivin was expressed in 5 cases (33.3%), p53 in 11 (73.3%), Bcl-2 in 4 (26.7%)
and Ki-67 was high in 6 lesions (40.0%) evidencing a variation in biomarker expression
pre and post-90Y-RE.

Changes of survivin, p53, Bcl-2 and Ki-67 in the 13 matched liver metastases pre-
and post-90Y-RE

In our series of liver biopsies, 13 patients had matched valuable tissues pre and
post-90Y-RE. As reported in Table 2, the 13 paired patients, included in biomarker analysis, were found to be representative
of the overall cohort of the 50 patients enrolled in the SITILO clinical trial with
no statistical differences between the groups for baseline parameters (sex, site of
primary tumors, number of metastases, liver involvement, performance status, bevacizumab
or cetuximab therapy). On the basis of this comparative analysis, we evaluated whether
survivin, p53, Bcl-2 and Ki-67 expression varied pre- and post-90Y-RE therapy in our series of 13 matched patients.

Table 2.Comparison of clinical variables between the overall series of patients and the series
with liver biopsies pre- and post-90Y-RE

As described in Figure 1 panel A, the IHC biomarker analysis in this subset of mCRC showed that post-90Y-RE there was a significant reduction in survivin positivity (from 92% to 54% of
samples; p = 0.06) and p53 nuclear accumulation (from 100% to 69%; p = 0.05) (Figure 1 panel B-a and B-b). Furthermore, we found a small, but significant, decrease in Bcl-2
positivity (from 46% to 31%; p = 0.05; Figure 1 panel B-c) and a limited, not significant, decrease in Ki-67 positivity (from 77%
to 61%).

In our series of 13 matched patients, 5 presented biomarker variations pre and post-90Y-RE therapy and 8 no biomarker variations. Of clinical interest, 6 of the latter
patients (75%) presented progression disease whereas all the 5 patients showing changes
in biomarker expression had partial response or stable disease (Figure 2, panel B). Nevertheless, the limited number of patients did not allow us to determine
whether these changes may really affect survival.

Discussion

Patients included in the present study were from a multicenter phase II clinical trial
which is the first prospective evaluation of 90Y-RE in CRC patients with liver metastases who failed previous oxaliplatinum and irinotecan
based chemotherapy regimen [10]. It has been widely reported that alterations in genes, as survivin, p53 and Bcl-2,
which regulate cell growth and apoptotic processes, are significantly associated to
an unfavourable clinical outcome in CRC patients [15]. In our series of 29 liver mCRC patients, we found that most tumors sampled prior
to 90Y-RE were p53, survivin, and Bcl-2 highly positive and presented a high Ki-67 proliferation
index. In contrast, we found a significant reduction in p53, survivin and Bcl-2 positive
expression in liver metastasis sampled two months post-90Y-RE. There was also a trend towards a reduction in cells with a high proliferative
index as measured by Ki-67. We have previously shown that colon cancers harboring
p53 nuclear accumulation, as assessed by the DO7 anti-p53 antibody, represent a subset
of tumors with a more aggressive clinical behaviour in patients with stage II tumors
as well as in young patients [13,16]. Furthermore, several studies have shown an increased incidence of p53 nuclear accumulation
in liver metastases in comparison to the primary tumor, hypothesizing a role for p53
in CRC liver metastatization. In particular, the presence of ≥ 3 liver metastases
identified a subset of patients with a very poor prognosis mainly when associated
to p53 mutations [17]. A number of studies have also shown that tumors that do not express detectable levels
of Bcl-2, but which exhibited nuclear accumulation of p53, were associated with the
shortest patient survival, while Bcl-2-positive and p53-negative tumors had the best
prognosis [12,17]. Studies conducted at our Institute showed that p53 positivity combined with Bcl-2
negativity and elevated Ki-67 score correlated with advanced tumor stage, poorly differentiated
tumors and increased probability of relapse. Also elevated survivin expression levels
in primary CRC are related to decreased survival [14,15]. In resected liver tumors, altered expression of survivin, p53, Ki-67 and, more recently,
KRAS mutations, have been shown to be independently predictive of hepatic recurrence
and poor survival [13,16,18]. It is recently reported that defective mismatch repair predicts resistance to 5-fluorouracil
(5FU) and KRAS mutation resistance to anti-EGFR antibody therapy [19]. Nevertheless, no predictive markers of RE efficacy in mCRC have been identified
up to now. In terms of the predictive response to radiotherapy, several studies have
linked epidermal growth factor receptor (EGFR) and vascular endothelial growth factor
(VEGF) expression to a lack of response to pre-operative radiotherapy in locally advanced
rectal cancer [19-21]. Neither p53, Ki-67 and survivin expression appear to be correlated to pre-operative
chemo-radiotherapy response and prognosis in locally advanced rectal cancer [22,23]. To date, however, no study has evaluated the predictive value of molecular markers
on radiosensitivity of CRC liver metastasis. In this context, our findings, although
in a very limited number of patients, may be clinically relevant.

The rapid changes of biomarkers observed in our series post-90Y-RE may be due to clonal selection or to epigenetic changes, not previously recorded
in this context. Such mechanisms are usually discussed in the context of cell adaption
to chemotherapy and evolving resistance. Radio-sensitivity of colorectal cancer cells
may be determined by p53 mutation [23,24], whereas there is no evidence that chemotherapy per se cause changes in the cellular
expression of p53 [25]. This is the first time that we have recorded a down-staging in p53 protein expression
after 90Y-RE.

It is likely that both disease progression and a prolonged prior chemotherapy affected
the efficacy and tolerability of 90Y-RE in the liver. In fact, mild manifestations of non-alcoholic fatty liver disease
(NAFLD) after 5FU [26], more serious non-alcoholic steatohepatitis after irinotecan and sinusoidal obstruction
syndrome (SOS) after oxaliplatin-based treatment [27] have been recorded. Using the same biomarkers as in our study, Panasiuk and colleagues
[28] showed that the intensification of inflammation in NAFLD may also impact on biomarker
expression in human hepatocytes with the induction of pro-apoptotic protein p53 and
the inhibition of anti-apoptotic Bcl-2.

There are clear limitations to our study, not least of which was the small patient
numbers and limited tissue sampling. Nevertheless, we believe that our findings merit
further investigation in prospective clinical trials. We are planning to evaluate
this biomarker panel in a phase II randomized trial on 2nd line treatment. KRAS mutated
CRC patients with unresectable liver metastasis will be randomized to receive systemic
therapy vs systemic therapy plus 90Y-RE. The combined assessment of survivin, p53 and Bcl-2 pre and post-90Y-RE therapy may improve our ability to predict outcomes in the treatment paradigm
of metastatic KRAS mutated CRC patients.

Competing interests

The authors declared that they have no competing interest.

Authors’ contributions

EM and CE carried out immunohistochemical staining and contributed in data acquirement
and interpretation. MC contributed to the study design, data interpretation and manuscript
drafting. LC, GP, FF, RG, EG performed liver biopsies pre and post radioembolization
in all the patients included in this study. IS was responsible for the database set
up and for the statistical analyses. RS was involved in the patient treatment with
ytttium-90 microspheres. MD evaluated the morphological features of liver biopsies
and revised all the slides submitted to immunohistochemical staining. CG and FI, RM
provided clinical and surgical data of the patients including treatment schedule and
follow up. MM were responsible for the study concept and design and for the interpretation
of results, helped in data discussion, critically revised the manuscript for important
intellectual content, and obtained funding for the study. All authors have read and
approved the manuscript.

Acknowledgements

We would to thank the patients for agreeing to participate in this study, which was
a collaboration of the Italian Society of Locoregional Therapies in Oncology (SITILO).
We would also like to thank Paolo Avetrani, PhD, and Rae Hobbs and Maria Assunta Fonsi
for their helpful contribution to this work.

The yttrium-90 resin microspheres were provided by Sirtex Medical Limited.